The A, B, and H antigens are a clinically significant blood group (Landsteiner, 1901; Mollison et al., 1987). These antigens are terminal immunodominant monosaccharides on erythrocyte membrane glycoconjugates (Harmening, 1989). High densities of these epitopes are present on erythrocyte membranes and antibodies bound to these antigens readily fix complement (Economidou et al., 1967; Romano and Mollison, 1987). Because these epitopes are ubiquitous in nature, immuno-potent and naturally occurring, complement fixing antibodies occur in individuals lacking these antigens, and transfusion of incompatible blood results in fatal hemolytic transfusion reactions (Fong et al., 1974; Schmidt, 1980).
Complex sugar chains in glycolipids and glycoproteins have often been implicated in the growth and development of eukaryotes (Wantanabe et al., 1976). In particular, complex sugar chains play an important part in the recognition of self in the immune system (Mollison, 1987). Glycosidases (both exoglycosidases and endoglycosidases) are enzymes which can modify carbohydrate membrane epitopes, thereby modulating the immune response (Goldstein et al., 1982). The .alpha.-N-acetyl-galactosaminidase from domestic chickens is an enzyme that degrades the human blood group A epitope (Hata et al., 1992). Degradation of the blood group A antigen produces the H antigen, also known as blood group O. Blood group O red blood cells are generally universally transfusable within the ABO blood group system.
The enzyme .alpha.-N-acetyl-galactosaminidase [EC 3.2.1.49] is a class of exoglycosidases that have been purified from both procaryotes and eucaryotes (McGuire et al., 1972; McDonald et al., 1972; Kadowaki et al., 1989; Itoh and Uda, 1984; Nakagawa et al., 1987; Kubo, 1989; Weissman et al., 1969; Weissman, 1972). Despite the use of this enzyme because of its ready availability from the livers of domesticated chickens, the use is limited because there are no published reports of preparations that have no detectable protease or other glycosidase activities with proven homogeneity utilizing commercially useful purification methods (Goldstein, 1984). Without a commercially viable method to provide enzymatic activity free of extraneous proteases and glycosidases, there is limited commercial value since the use of a nonhomogeneous enzyme preparation has the potential to damage erythrocyte membranes leading to poor in vivo viability. It would be particularly advantageous to be able to isolate commercially significant amounts of the enzyme from chicken livers because of the low cost of the starting material or to have available cDNA for the enzyme and associated vectors and host cells to enable production of the enzyme in culture.
U.S. Pat. No. 4,330,619, issued May 18, 1982; 4,427,777, issued Jan. 24, 1984; and U.S. Pat. No. 4,609,627, issued Sep. 2, 1986, all to Goldstein, relate to the enzymatic conversion of certain erythrocytes to type O erythrocytes. Since type O erythrocytes can be safely transfused into type A, type B, type A,B recipients, as well as O recipients, type O erythrocytes have significant value in transfusion therapy. The above-mentioned U.S. Pat. No. 4,609,627 discloses the conversion of certain sub-type A and A,B erythrocytes to type O erythrocytes utilizing an .alpha.-N-acetyl-galactosaminidase fraction from fresh chicken livers. The patent also discusses the significant potential of such enzymes to be used in the conversion of type A.sub.2 erythrocytes to type O erythrocytes.
The Gallus enzyme more specifically has significance because of its specificity for the terminal N-acetyl-.alpha.-D-galactosaminyl residues of the human blood group A antigen. Hydrolysis of the terminal N-acetyl-.alpha.-D-galactosaminyl monosaccharide results in the less immunogenic type O erythrocyte blood group (Goldstein, 1989). As stated above, the use of a purified Gallus enzyme having significantly increased activity can be an important contributor to enlarging the available compatible blood supply for transfusion therapy.
The purification method for isolating .alpha.-N-acetyl-galactosaminidase from chicken livers disclosed in the U.S. Pat. No. 4,609,627 patent requires dehydrating the tissue, extensive protein precipitation steps, extension column chromatography steps, as set forth in FIG. 1, and most importantly, the yields are low compared to the amount of starting material.
The published application (WO 99409121) of Goldstein and Hurst provides a second purification protocol as set forth in FIG. 2. However, this protocol also requires extensive handling of the tissue, dialysis and more particularly, extensive column chromatography. This type of protocol is expensive, time-consuming, labor intensive and most importantly, the yields are low compared to the amount of starting material. More particularly, the procedure requires the homogenization of 5 pounds (2,270 g) of chicken liver with only 1.8 liter of buffer. This is a 56% w/v homogenate which is a thick, viscous slurry which is inefficiently resolved by filtration or centrifugation. Furthermore, this procedure requires three dialysis steps and two ammonium sulfate precipitations as well as extensive column chromatography.
Modification of isolation procedures to commercial scale cannot always be accomplished by simply increasing the amount of starting material. This does not always increase the yield proportionally. Problems of non-homogeneous product can occur, particularly if steps are eliminated. Further, contamination of the product with proteases and contamination of the column resins often occurs as the amount of material being processed increases.
The present invention provides an improved method of providing isolated and purified glycosidases. The present invention also provides a cDNA for .alpha.-N-acetyl-galactosaminidase from Gallus domesticus and vectors and host cells containing the cDNA.